Cell cycle checkpoints are implemented to safeguard our genome and the deregulation of which contributes to the pathogenesis of human cancers. Hence, it is of paramount importance to discover and interrogate novel key constituents of the mammalian DNA damage response network. Among G1-, S-, G2- and M-phase checkpoints, genetic studies indicate the essence of an intact S-phase checkpoint in maintaining genome integrity. Although basic framework of the S-phase checkpoint in multi-cellular organisms has been outlined, the mechanistic details remain to be elucidated. Human chromosome band 11q23 translocation disrupting the MLL gene results in poor prognostic leukemias that carry pathognomonic MLL fusions. MLL is a transcription co-activator that is best known to maintain HOX gene expression. The importance of HOXA gene deregulation in MLL leukemogenesis has been intensively investigated. However, physiological murine MLL leukemia knockin models indicated that MLL fusion-induced HOXA gene aberration alone is insufficient to initiate MLL leukemia. Therefore, further dysregulation must exit and contribute to the ultimate leukemia phenotype. Our recent studies demonstrated a close relationship between MLL and the regulation of mammalian cell cycle. MLL not only assists in the G1/S and G2/M phase transition during a normal cell division cycle but also executes the S-phase checkpoint upon DNA damage. We found that (1) MLL functions as a key effector of ATR-mediated S-phase checkpoint response, (2) activated ATR phosphorylates and thus stabilizes MLL, (3) upon checkpoint activation MLL accumulates at the late replication origin, methylates histone H3K4, and thus delays DNA replication, (4) MLL deficient cells exhibit defects in the S-phase checkpoint response, and (5) MLL fusions work as dominant negative mutants that compromise the integrity of S-phase checkpoint. Here we will determine the mechanisms by which MLL executes the S phase checkpoint response and examine whether and to what extent an S-phase checkpoint dysfunction contributes to MLL leukemogenesis. Our proposal connects MLL/MLL fusions to the S-phase checkpoint response network, which not only provides novel insights into the mammalian cell cycle checkpoint control but also shed light on the pathogenesis of poor prognostic human leukemias.